Project Summary Multiple lines of evidence support wide-spread structural and functional maladaptations in brain circuits as a contributing factor promoting chronic pain. Dysregulation of descending pain modulatory circuits have been demonstrated in chronic pain patients. However, our understanding of the molecular mechanisms and brain circuits that underlie inhibition or facilitation of pain under normal and pathological conditions remains limited. Opioid analgesics engage these brain circuits, but non-addictive options to treat pain are urgently needed. In this proposal, we will investigate pain modulatory mechanisms in the amygdala, a structure involved in integration of pain-related sensory inputs and emotional processing. We will test the hypothesis that functionally opposing endogenous mu (MOR) and kappa (KOR) opioid circuits in the central nucleus of the amygdala (CeA) inhibit or promote, respectively, pain behavior. Bidirectional descending pain modulatory circuits have been characterized most extensively in the rostral ventromedial medulla (RVM). However, little is known about descending modulatory circuits above the brainstem. In the RVM, pro-nociceptive and anti-nociceptive functions are mediated by MOR-expressing ?pain ON? cells and KOR-expressing ?pain OFF? cells. We propose that similar functional organization with MOR- and KOR-expressing cells serving opponent roles in sensory and/or affective pain behavior also exists in the CeA. Importantly, the reciprocal function of the MOR and KOR circuits may represent a general organizational principle for pain modulation in other brain areas. We will use transgenic mouse models in combination with optogenetic and chemogenetic approaches and anterograde and retrograde tracing for the cell type-specific and projection-specific analysis of MOR and KOR circuits in the CeA and their contribution to pain behaviors in a neuropathic pain model (spinal nerve ligation). Specific Aim 1 will determine the anatomical characteristics of KOR and MOR cells in the CeA. Specific Aim 2 will explore the synaptic and cellular effects of KOR and MOR activation or inhibition and their interaction. Specific Aim 3 will assess the role of MOR and KOR circuits in pain behaviors using reflexive responses (mechanical and thermal thresholds) as well as complex behaviors related to affective and motivational qualities of pain (vocalization, conditioned place preference/avoidance). Eletrophysiological and behavioral outcomes will be assessed with cell specific manipulations under control conditions and in a neuropathic pain model in both male and female mice allowing the determination of possible sex differences in the amygdala circuit. The proposed studies will identify a previously unknown KOR-mediated pro-nociceptive amygdala circuit and its relationship to MOR-mediated anti-nociceptive mechanisms. Impaired MOR signaling and/or increased KOR signaling in the CeA may result in an inhibition-excitation imbalance of descending modulation to promote chronic pain. Importantly, the opposing MOR-KOR function in the CeA would provide the conceptual basis for the development of non-addictive therapies for chronic pain.